We demonstrated for the first time that the biosynthesis of fatty acids is an important metabolic pathway in weight-stable humans. Using a new experimental approach to measure fatty acid synthesis in vivo, we determined whether a eucaloric, low fat, high carbohydrate diet increased fatty acid synthesis. Normal volunteers consumed low fat liquid formula diets (% of calories as fat and 75% as glucose polymers, n=7) or high fat diets (40% of calories as fat and 45% as glucose polymers, n=3) for 25 days at the Rockefeller University GCRC. The fatty acid composition of each diet was matched to the composition of each subject's adipose tissue and compared to the composition of VLDL triglyceride. By day 10, VLDL triglyceride was markedly enriched in the saturated fatty acid, palmitate, and deficient in the essential fatty acid, linoleate, in all subjects on the low fat diet. Newly synthesized fatty acids accounted for 44 *10% of the VLDL triglyceride. Mass isotopomer distribution analysis of palmitate labeled with intravenously infused 13C-acetate confirmed that increased palmitate synthesis was the likely cause for the accumulation of triglyceride palmitate and " dilution" of linoleate. In contrast, there was minimal fatty acid synthesis on the high fat diet. Thus, the dietary substitution of simple carbohydrate for fat stimulated fatty acid synthesis and the plasma accumulation of palmitate-enriched, linoleate- deficient triglyceride. Such changes in plasma lipids could have adverse effects on the cardiovascular system, including alterations in the atherogenicity of lipoproteins, blood coagulation, and membrane receptor function.